Diazabenzo[de] anthracen-3-one compounds and methods for inhibiting parp

ABSTRACT

The present invention relates to diazabenzo[de]anthracen- 3 .-onei compounds which inhibit poly(ADP-ribose) polymerase (“PARP”), compositions containing these compounds and methods for using these PARP inhibitors to treat, prevent and/or ameliorate the effects of the conditions described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 11/814,238, filed on Mar. 21, 2008. U.S.application Ser. No. 11/814,238 is a national stage of PCT/US06/01729,filed on Jan. 19, 2006, which claims the benefit of U.S. application60/644,584, filed on Jan. 19, 2005 and U.S. application 60/712,140,filed Aug. 30, 2005. The entire contents of these applications are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to diazabenzo[de]anthracen-3-one compoundswhich inhibit poly(ADP-ribose) polymerase (“PARP”), compositionscontaining these compounds and methods for using these PARP inhibitorsto treat, prevent and/or ameliorate the effects of the conditionsdescribed herein.

BACKGROUND

PARP (EC 2.4.2.30), also known as PARS (for poly(ADP-ribose)synthetase), or ADPRT (for NAD:protein (ADP-ribosyl) transferase(polymerising)) is a major nuclear protein of 116 kDa. It is mainlypresent in almost all eukaryotes. The enzyme synthesizespoly(ADP-ribose), a branched polymer that can consist of over 200ADP-ribose units from NAD. The protein acceptors of poly(ADP-ribose) aredirectly or indirectly involved in maintaining DNA integrity. TheyInclude histones, topoisomerases, DNA and RNA polymerases, DNA ligases,and Ca²⁺- and Mg⁺-dependent endonucleases.

PARP protein is expressed at a high level in many tissues, most notablyin the immune system, heart, brain and germ-line cells. Under normalphysiological conditions, there is minimal PARP activity. However, DNAdamage causes an immediate activation of PARP by up to 500-fold. Amongthe many functions attributed to PARP is its major role in facilitatingDNA repair by ADP-ribosylation and therefore coordinating a number ofDNA repair proteins. As a result of PARP activation, NAD levelssignificantly decline. While many endogenous and exogenous agents havebeen shown to damage DNA and activate PARP, peroxynitrite, formed from acombination of nitric oxide (NO) and superoxide, appears to be a mainperpetrator responsible for various reported disease conditions in vivo,e.g., during shock, stroke and inflammation.

It is also known that PARP inhibitors, such as 3-amino benzamide, affectDNA repair generally in response, for example, to hydrogen peroxide orgamma-radiation, Cristovao et al., “Effect of a Poly(ADP-Ribose)Polymerase Inhibitor on DNA Breakage and Cytotoxicity Induced byHydrogen Peroxide and γ-Radiation,” Terato., Carcino., and Muta.,16:219-27 (1996). Specifically, Cristovao et al. observed aPARP-dependent recovery of DNA strand breaks in leukocytes treated withhydrogen peroxide.

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) plays a keyrole in facilitating base excision repair and other cellular processes.It has been proposed that PARP-1 acts as a molecular DNA nick sensor,detecting DNA single-strand breaks and recruiting the appropriate repairenzymes. PARP-1 binds to DNA strand breaks via two zinc fingers in theamino-terminal DNA binding domain of the enzyme, its activity beingdependent on DNA binding. The enzyme acts as a homodimer catalyzing thetransfer of ADP-ribose from the substrate NAD+ to acceptor proteins,including PARP-1 itself. Extensive negatively charged polymers of PARare thereby formed, causing electrostatic repulsion of DNA strands andchromatin proteins, the latter allowing base excision repair complexesaccess to the damaged strand and subsequent DNA repair. After initialactivation by a strand break, PARP-1 is released from the DNA, thepolymer degraded by PAR glycohydrolase, and the PARP-1 enzyme is thenavailable for a further round of DNA binding and activation. Plummer, etal., 11 (9) Clin. Cancer Res. 3402 (2005).

PARP inhibitors have been reported to be effective, as synergists orpotentiators, in radiosensitizing hypoxic tumor cells. PARP inhibitorshave also been reported to be effective as synergists in preventingtumor cells from recovering from potentially lethal damage of DNA afterradiation therapy, presumably by their ability to prevent DNA repair.US. Pat. Nos. 5,032,617; 5,215,738; and 5,041,653.

There is considerable interest in the development of PARP inhibitors asboth chemopotentiators and radiopotentiators for use in cancer therapyand to limit cellular damage after ischemia or endotoxic stress. Inparticular, potentiation of temozolomide cytotoxicity observed inpreclinical studies with potent PARP-1 inhibitors reflects inhibition ofbase excision repair and subsequent cytotoxicity due to incompleteprocessing of N⁷-methylguanine and H³-methyladenine. There is now a bodyof preclinical data demonstrating that the cytotoxicity of temozolomideis potentiated by coadministration of a PARP inhibitor either in vitroor in vivo. Plummer, et al., Clin. Cancer Res., 11 (9), 3402 (2005).

Temozolomide, a DNA methylating agent, induces DNA damage, which isrepaired by O⁶-alkylguanine alkyltransferase (ATase) andpoly(ADP-ribose) polymerase-1 (PARP-1 )-dependent base excision repair.Temozolomide is an orally available monofunctional DNA alkylating agentused to treat gliomas and malignant melanoma. Temozolomide is rapidlyabsorbed and undergoes spontaneous breakdown to form the activemonomethyl triazene, 5-(3-methyl-1-triazeno)imidazole-4-carboxamide.Monomethyl triazene forms several DNA methylation products, thepredominate species being N⁷-methylguanine (70%), N³-methyladenine (9%),and O⁶-methylguanine (5%). Unless repaired by O⁶-alkylguaninealkyltransferase, O⁶-methylguanine is cytotoxic due to mispairing withthymine during DNA replication. This mispairing is recognized on thedaughter strand by mismatch repair proteins and the thymine excised.However, unless the original O⁶-methylguanine nucleotide in the parentstrand is repaired by ATase-mediated removal of the methyl adduct,thymine can he reinserted. Repetitive futile rounds of thymine excisionand incorporation opposite an unrepaired O⁶-methylguanine nucleotidecauses a state of persistent strand breakage and the MutS branch ofmismatch repair system signals G2-M cell cycle arrest and the initiationof apoptosis. The quantitatively more important N⁷-methylguanine andN³-methyladenine nucleotide alkylation products formed by temozolomideare rapidly repaired by base excision repair. Plummer, et al., Clin.Cancer Res., 11 (9), 3402 (2005).

Chemosensitization by PARP inhibitors is not limited to temozolomide.Cytotoxic drugs, generally, or radiation can induce activation of PARP-1and it has been demonstrated that inhibitors of PARP-1 can potentiatethe DNA damaging and cytotoxic effects of chemotherapy and irradiation.Kock, et al., 45 J. Med Chem. 4901 (2002). PARP-1 mediated DNA repair inresponse to DNA damaging agents represents a-mechanism for drugresistance in tumors, and inhibition of this enzyme has been shown toenhance the activity of ionizing radiation and several cytotoxicantitumor agents, including temozolomide and topotecan. Suto et al., inU.S. Pat. No. 5,177,075, disclose several isoquinolines used forenhancing the lethal effects of ionizing radiation or chemotherapeaticagents on tumor cells. Weltin et al., “Effect of 6(5H)-Phenanthridinone,an Inhibitor of Poly(ADP-ribose) Polymerase, on Cultured Tumor Cells”,Oncol. Res., 6:9, 399-403 (1994) disclose the inhibition of PARPactivity, reduced proliferation of tumor cells, and a marked synergisticeffect when tumor cells are co-treated with an alkylating drug. PARP-1is thus a potentially important therapeutic target for enhancingDNA-damaging cancer therapies.

Large numbers of known PARP inhibitors have been described in Banasik etal., “Specific Inhibitors of Poly(ADP-Ribose) Synthetase andMono(ADP-Ribosyl)-Transferase”, J. Biol. Chem., 267:3, 1569-75 (1992),and in Banasik et al., “Inhibitors and Activators of ADP-RibosylationReactions”, Molec. Cell. Biochem., 138, 185-97 (1994). However,effective use of these PARP inhibitors, in the ways discussed above, hasbeen limited by the concurrent production of unwanted side-effects. SeeMilam et al., “Inhibitors of Poly(Adenosine Diphosphate-Ribose)Synthesis; Effect on Other Metabolic Processes,” Science, 223, 589-91(1984).

In addition to the above, PARP Inhibitors have been disclosed anddescribed in the following international patent applications: WO00/42040; WO 00/39070; WO 00/39104; WO 99/11623; WO 99/11628; WO99/11622; WO 99/59975; WO 99/11644; WO 99/11945; WO 99/11649; and WO99/59973. A comprehensive review of the state of the art has beenpublished by Li and Zhang in IDrugs 2001, 4 (7): 804-812 (PharmaPressLtd ISSN 1369-7056).

The ability of PARP-inhibitors to potentiate the lethality of cytotoxicagents, whether by radiosensitizing tumor cells to ionizing radiation,or by chemosensitizing tumor cells to the cytotoxic effects ofchemotherapeutic agents has been reported in, inter alia,. U.S.2002/0028815; U.S. 2003/0134843; U.S. 2004/0067949; White A W, et al.,14 Bioorg. & Med. Chem Letts. 2433 (2004); Canon Koch S S, et al., 45 J.Med. Chem. 4961 (2002); Skalitsky D J, et al., 46J. Med. Chem. 210(2003); Farmer H, et al., 434 Nature 917 (14 Apr. 2005); Plummer E R, etal., 11 (9) Clin. Cancer Res. 3402 (2005); Tikhe J G, et al., 47 J. Med.Chem. 5467 (2004); Griffin R. J., et al., WO 98/33802; and Helleday T,et al., WO 2005/012305.

The induction of peripheral neuropathy is a common factor in limitingtherapy with chemotherapeutic drugs. Quasthoff and Hartung, J.Neurology, 249, 9-17 (2002). Chemotherapy induced neuropathy is aside-effect encountered following the use of many of the conventional(e.g., Taxol, vincritine, cisplatin) and newer chemotherapies (egvelcade, epothilone). Depending on the substance used, a pure sensoryand painful neuropathy (with cisplatin, oxaliplatin, carboplatin) or amixed sensorimotor neuropathy with or without involvement of theautonomic nervous system (with vincristine, taxol, suramin) can ensue.Neurotoxicity depends on the total cumulative dose and the type of drugused. In individual cases neuropathy can evolve even after a single drugapplication. The recovery from symptoms is often incomplete and a longperiod of regeneration is required to restore function. Up to now, nodrug is available to reliably prevent or cure chemotherapy-inducedneuropathy.

There continues to be a need for effective and potent PARP inhibitorswhich enhance the lethal effects of ionizing radiation and/orchemotherapeutic agents on tumor cells while producing minimalside-effects.

SUMMARY OP THE INVENTION

The present invention provides diazabenzo[de]anthracen-3-one compoundswhich inhibit poly(ADP-ribose) polymerase (“PARP”), compositionscontaining these compounds and methods for using these PARP inhibitorsto treat, prevent and/or ameliorate the effects of the conditionsdescribed herein.

The present invention also provides a diazabenzo[de]anthracen-3-onecompound selected from the following Group I compounds:

pharmaceutically acceptable salts, hydrates, esters, solvates, andmixtures thereof.

The present invention also relates to a pharmaceutical compositioncomprising (i) a therapeutically effective amount of a compound of GroupI and (ii) a pharmaceutically acceptable carrier.

The present invention provides compounds which inhibit the in vitroand/or in vivo polymerase activity of poly(ADP-ribose) polymerase(PARP), and compositions containing the disclosed compounds.

The present invention provides methods to inhibit, limit and/or controlthe in vitro and/or in vivo polymerase activity of poly(ADP-ribose)polymerase (PARP) in solutions, cells, tissues, organs or organ systems.In one embodiment, the present invention provides methods of limiting orinhibiting PARP activity in a mammal, such as a human, either locally ofsystemically.

The present invention provides methods to treat and/or prevent diseases,syndromes and/or conditions exacerbated by or involving the increasedgeneration of PARP. These methods involve application or administrationof the compounds of the present invention to cells, tissues, organs ororgan systems of a person in need of such treatment or prevention.

In another embodiment the compounds and compositions of the presentinvention can be used to treat or prevent cell damage or death due tonecrosis or apoptosis, cerebral ischemia and reperfusion injury orneurodegenerative diseases in an animal, such as a human.

In another embodiment the compounds and compositions of the presentinvention can be used to extend the lifespan and proliferative capacityof cells and thus can be used to treat or prevent diseases associatedtherewith.

In another embodiment, the present invention provides methods oftreating of preventing or ameliorating the effect of cancer and/or toradiosensitive tumor cells or hypoxic tumor cells to render the tumorcells more susceptible to radiation therapy and thereby to prevent thetumor cells from recovering from potentially lethal damage of DNA afterradiation therapy. A method of this embodiment is directed tospecifically and preferentially radiosensitizing tumor cells renderingthe tumor cells more susceptible to radiation therapy than non-tumorcells.

The present invention also provides diazabenzo[de]anthracen-3-onecompounds of Group I to treat, prevent and/or ameliorate the effects ofcancers by potentiating the cytotoxic effects of ionizing radiationand/or chemotherapeutic agents on tumor cells.

In one embodiment, the invention provides a chemosensitization methodfor treating cancers and or tumors comprising contacting the tumor orcancer cells with a cytotoxicity-potentlatingdiazabenzo[de]anthracen-3one compound of Group I and further contactingthe tumor or cancer cells with an anticancer agent.

The present invention provides a chemosensitization method for treatingcancers in a mammal, particularly a human, comprising administering tothe mammal a diazabenzo[de]anthracen-3-one compound selected from GroupI.

In one embodiment of the invention, the compound for use in thechemosensitization method of the invention is

In another embodiment the present invention provides achemosensitization method wherein a first dose of at least one compoundof Group I is administered singly or repeatedly to a patient in needthereof, and wherein subsequently a second dose of at least onechemotherapeutic agent is administered singly or repeatedly to saidpatient after a time period to provide an effective amount ofchemosensitization.

In another embodiment the present invention provides a pharmaceuticalformulation comprising the chemosensitizing diazabenzo[de]anthracen-3-one derivative in a form selected from the group consisting ofpharmaceutically acceptable free bases, salts, hydrates, esters,solvates, prodrugs, metabolites, stereoisomers, and mixtures thereof.According to a further embodiment, the pharmaceutical formulationfurther comprises a pharmaceutically acceptable carrier and, optionally,a chemotherapeutic agent. Non-limiting examples of such chemotherapeuticagents are recited below.

According to another embodiment of the invention, the chemosensitizingcompound and the chemotherapeutic agent are administered essentiallysimultaneously.

According to another embodiment of the invention, the chemotherapeuticagent is selected from the group consisting of temozolomide, adriamycin,camptothecin, carboplatin, cisplatin, daunorubicin, docetaxeLdoxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan,paclitaxel, a taxoid, dactinomycin, danorubicin, 4′-deoxydoxorubicin,bleomycin, pilcamycin, mitomycin, neomycin and gentamycin, etoposide,4-OH cyclophosphamide, a platinum coordination complex, topotecan,therapeutically effective analogs and derivatives of the same, andmixtures thereof. According to a preferred aspect, the cherootherapeuticagent is temozolomide.

In one embodiment the present invention provides a pharmaceuticalcomposition comprising a chemosensitizing effective amount of at leastone diazabenzo[de]anthracen-3-one compound selected from Group I. Inanother aspect, the pharmaceutical composition comprises

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. I demonstrates that in mice bearing B16 melanoma, the mean survivaltime of the groups treated with compound 4i+TMZ combination wassignificantly higher than that observed in animals receiving TMZ assingle agent.

FIG. II shows that the combination treatment of Compound 4i+TMZsignificantly reduced the growth of B16 melanoma (P<0.01 from day 9 today 23, vs TMZ alone).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The present invention relates to the use of compounds of the presentinvention in the preparation of a medicament for the treatment of anydisease or disorder in an animal or mammal described herein.

As used herein, “alkyl” means a branched or unbranched saturatedhydrocarbon chain comprising a designated number of carbon atoms. Forexample, C₁-C₆ straight or branched alkyl hydrocarbon chain contains 1to 6 carbon atoms, and includes but is not limited to substituents suchas methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl,n-pentyl, n-hexyl, and the like, unless otherwise indicated.

“Alkenyl” means a branched or unbranched unsaturated hydrocarbon chaincomprising a designated number of carbon atoms. For example, C₂-C₆straight or branched alkenyl hydrocarbon chain contains 2 to 6 carbonatoms having at least one double bond, and. includes but is not limitedto substituents, such as ethcayl, propenyl, isopropenyl, batenyl,Iso-butenyl, tert-botenyi, n-pentenyl, n-hexenyl, and the like, unlessotherwise indicated.

“Alkoxy”, means the group —OR wherein R is alkyl as herein defined. Rcan also be a branched or unbranched saturated hydrocarbon chaincontaining 1 to 6 carbon atoms.

“Cyclo”, used herein as a prefix, refers to a structure characterized bya closed ring.

“Halo” means at least one fluoro, chloro, promo, or iodo moiety, unlessotherwise indicated.

“Amino” compounds include amine (NH₂) as well as substituted amino.

“Ar”, “aryl” or “heteroaryl” means a moiety which is substituted orunsubstituted, especially a cyclic or fused cyclic ring and includes amono-, bi-, or tricyclic, carbo- or heterocyclic ring, wherein the ringis either unsubstituted or substituted in one to five position(s) withhalo, haloalkyl, hydroxyl, niiro, trifluoromethyl, C₁-C₆ straight orbranched chain alkyl, C₂-C₆ straight or branched chain alkenyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy, amino, thiocarbonyl,ester, thioester, cyano, imino, alkylamino, aminoalkyl, sulfhydryl,thioalkyl, and sulfonyl; wherein the individual ring sizes are 5-8members; wherein the heterocyclic ring contains 1-4 heteroatom(s)selected from the group consisting of O, N, or S; wherein aromatic ortertiary alkyl amines are optionally oxidized to a correspondingN-oxide. Heteroaryls may be attached to other rings or substitutedthrough the heteroatom and/or carbon atom of the ring. Aryl orheteroaryl moieties include but are not limited to phenyl, benzyl,naphthyl, pyrrolyl, pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl,quinolinyl, isoquinolinyl, furyl, thiophenyl, imidazolyl, oxazolyl,thiazolyl, pyrazolyl and thieny.

“Phenyl” includes all possible isomeric phenyl radicals, optionallymonosubstituted or multi-substituted with substitueuts selected from thegroup consisting of amino, trifluoromethyl, C₁-C₆ straight or branchedchain alkyl, C₂-C₆ straight or branched chain alkenyl, carbonyl,thiocarbonyl, ester, thioester, alkoxy, alkenoxy, cyano, nitro, imino,alkylamino, aminoalkyl, sulfhydryl, thioalkyl, sulfonyl, hydroxy, halo,haloalkyl, NR₂ wherein R₂ is selected from the group consisting ofhydrogen, (C₁-C₆)-straight or branched chain alkyl, (C₃-C₆) straight orbranched chain alkenyl or alkynyl, and (C₁-C₄) bridging alkyl whereinsaid bridging alkyl forms a heterocyclic ring starting with the nitrogenof NR₁ and ending with one of the carbon atoms of said alkyl or alkenylchain, and wherein said heterocyclic ring is optionally fused to an Argroup.

Cycloalkyl optionally containing at least one heteroatom includessaturated C₃-C₈ rings, such as C₅ or C₆ rings, wherein at 1-4heteroatoms selected from O, N or S may be optionally substituted for acarbon atom of the ring. Cycloalkyls optionally containing at least oneheteroatom, as described above, may be substituted by or fused to atleast one 5 or 6 membered aryl of heteroaryl. Other cycloalkylscontaining a heteroatom include pyrrolidinyl, imidazolidinyl,pyrazolidinyl, piperidinyl, piperazinyl, morpholino and thiomorpholino.

The term “neurodegenerative diseases” includes, but is not limited toAlzheimer's disease. Parkinson's disease and Huntington's disease.

The term “nervous insult” refers to any damage to nervous tissue and anydisability or death resulting therefrom. The cause of nervous insult maybe metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, andincludes without limitation, ischemia, hypoxia, cerebrovascularaccident, trauma, surgery, pressure, mass effect, hemmorrhage,radiation, vasospasm, neurodegenerative disease, infection, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS), myelination/demyelinationprocess, epilepsy, cognitive disorder, glutamate abnormality andsecondary effects thereof.

The term “neuroprotective” refers to the effect of reducing, arrestingor ameliorating nervous insult, and protecting, resuscitating, orreviving nervous tissue that has suffered nervous insult.

The term “preventing neurodegeneration” includes the ability to preventa neurodegenerative disease or preventing further neurodegeneration inpatients who are already suffering from or have symptoms of aneurodegenerative disease.

The term “treating” refers to:

-   -   (i) preventing a disease, disorder or condition from occurring        in an animal that may be predisposed to the disease, disorder        and/or condition, but has not yet been diagnosed as having it;    -   (ii) inhibiting the disease, disorder or condition, i.e.,        arresting its development; and    -   (iii) relieving the disease, disorder or condition, i.e.,        causing regression of the disease, disorder and/or condition.

The term “neural tissue damage resulting from ischemia and reperfusioninjury and neurodegenerative diseases” includes damage due toneurotoxicity, such as seen in vascular stroke and global and focalischemia.

The term “ischemia” relates to localized tissue anemia due toobstruction of the inflow of arterial blood. Global ischemia occursunder conditions in which blood flow to the entire brain ceases for aperiod of time, such as may result from cardiac arrest. Focal ischemiaoccurs under conditions in which a portion of the brain is deprived ofits normal blood supply, such as may result from thromboembolyticocclusion of a cerebral vessel, traumatic head injury, edema, and braintumors.

The term “cardiovascular disease” relates to myocardial infarction,angina pectoris, vascular or myocardial ischemia, and related conditionsas would be known by those of skill in the art which involve dysfunctionof or tissue damage to the heart or vasculature, and especially, but notlimited to, tissue damage related to PARP activation.

The term “radiosensitizer”, as used herein, is defined as a molecule,such as a low molecular weight molecule, administered to animals intherapeutically effective amounts to increase the sensitivity of theceils to be radiosensitized to electromagnetic radiation and/or topromote the treatment of diseases which are treatable withelectromagnetic radiation. Diseases which are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells. Electromagnetic radiationtreatment of other diseases not listed herein are also contemplated bythe present invention. The terms “electromagnetic radiation” and“radiation” as used herein includes, but is not limited to, radiationhaving the wavelength of 10⁻²⁰ to 10⁰ meters. Preferred embodiments ofthe present invention employ the electromagnetic radiation of:gamma-radiation (10⁻²⁰ to 10⁻¹³ m) x-ray radiation (10⁻¹¹ to 10⁻⁹ m),ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm).infrared radiation (700 nm to 1.0 mm), or microwave radiation (1 mm to30 cm).

The term “chemosensitizer”, as used herein, refers to the ability of thecompounds of the invention to potentiate the antitumors activity ofchemotherapeutic agents. Such chemosensitization is useful, for example,to increase the tumor growth-retarding or -arresting effect of a givendose of a chemotherapeutic agent, or to improve the side-effect profileof a chemotherapeutic agent by allowing for reductions in its dose whilemaintaining its antitumoral efficacy.

Pharmaceutical Uses of the Invention

The present invention provides compounds, methods and pharmaceuticalcompositions for inhibiting the nuclear enzyme poly(adenosine5′-diphospho-ribose) polymerase [“poly(ADP-ribose) polymerase” or“PARP”, which is also referred to as ADPRT (NAD:protein (ADP-ribosyltransferase (polymersing)), pADPRT (poly(ADP-ribose) transferase) andPARS (poly(ADP-ribose) synthetase). Moreover, the present inventionprovides methods of using PARP inhibitors of the invention to preventand/or treat tissue damage resulting from cell damage or death due tonecrosis or apoptosis; neural tissue damage resulting from, for example,ischemia and reperfusion injury, such as cerebral ischemic stroke, headtrauma or spinal cord injury; neurological disorders andneurodegenerative diseases, such as, for example, Parkinson's orAlzheimer's diseases and multiple sclerosis; to prevent or treatvascular stroke; to treat or prevent cardiovascular disorders, such as,for example, myocardial infarction; to treat other conditions and/ordisorders such as, for example, age-related muscular degeneration, AIDSand other immune senescence diseases, arthritis, atherosclerosis, ataxiatelangiectasia, cachexia, cancer, degenerative diseases of skeletalmuscle involving replicative senescence, diabetes (such as diabetesmellitus), inflammatory bowel disorders (such as colitis and Crohn'sdisease), acute pancreatitis, mucositis, hemorrhagic shock, splanchnicartery occlusion shock, multiple organ failure (such as involving any ofthe kidney, liver, renal, pulmonary, retinal, pancreatic and/or skeltalmuscle systems), acute autoimmune thyroiditis, muscular dystrophy,osteoarthritis, osteoporosis, chronic and acute pain (such asneuropathic pain), renal failure, retinal ischemia, septic shock (suchas endotoxic shock), local and/or remote endothelial cell dysfunction(such are recognized by endo-dependent relaxant responses andup-regulation of adhesion molecules), inflammation and skin aging; toextend the lifespan and proliferative capacity of cells, such as, forexample, as general mediators in the generation of oxidants,proinflammatory mediators and/or cytokines, and general mediators ofleukocyte infiltration, calcium ion overload, phospholipid peroxidaion,impaired nitric oxide metabolism and/or reduced ATP production; to altergene expression of senescent cells; or to radiosensitize hypoxic tumorcells.

The compounds of the present invention can treat or prevent tissuedamage resulting from cell damage or death due to necrosis or apoptosis;can ameliorate neural or cardiovascular tissue damage, including thatfollowing focal ischemia, myocardial infarction, and reperfusion injury;can treat various diseases and conditions caused or exacerbated by PARPactivity; can extend or increase the lifespan or proliferative capacityof ceils; can alter the gene expression of senescent cells; and canradiosensitize cells. Generally, inhibition of PARP activity spares thecells from energy loss, preventing, in the case of neural cells,irreversible depolarization of the neurons, and thus, providesneuroprotection. While not being bound to any one particular theory, itis thought that PARP activation may play a common role in still otherexcitotoxic mechanisms, perhaps as yet undiscovered, in addition to theproduction of free radicals and NO.

For the foregoing reasons, the present invention further relates to amethod of administering a therapeutically effective amount of theabove-identified compounds in an amount sufficient to inhibit PARPactivity, to treat or prevent tissue damage resulting from cell damageor death due to necrosis or apoptosis, to effect a neuronal activity notmediated by NMDA toxicity, to effect a neuronal activity mediated byNMDA toxicity, to treat neural tissue damage resulting from ischemia andreperfusion injury, neurological disorders and neurodegenerativediseases; to prevent or treat vascular stroke; to treat or preventcardiovascular disorders; to treat other conditions and/or disorderssuch as age-related muscular-degeneration, AIDS and other immunesenescence diseases, arthritis, atherosclerosis, ataxia telangiectasia,cachexia, cancer, degenerative diseases of skeletal muscle involvingreplicative senescence, diabetes, head trauma, immune senescence,inflammatory bowel disorders (such as colitis and Crohn's disease),muscular dystrophy, osteoarthritis, osteoporosis, chronic and/or acutepain (such as neuropathic pain), renal failure, retinal ischemia, septicshock (such as endotoxic shock), and skin aging; to extend the lifespanand proliferative capacity of cells; to alter gene expression ofsenescent ceils; or to radiosensitize hypoxic tumor cells. The presentinvention also relates to treating diseases and conditions in an animalwhich comprises administering to said animal a therapeutically effectiveamount of the above-identified compounds.

The present invention relates to a method of treating, preventing orinhibiting a neurological disorder in an animal, which comprisesadministering to said animal a therapeutically effective amount of theabove-identified compounds. In a another embodiment, the neurologicaldisorder is selected from the group consisting of peripheral neuropathycaused by physical injury or disease state, traumatic brain injury,physical damage to the spinal cord, stroke associated with brain damage,focal ischemia, global ischemia, reperfusion injury, demyelinatingdisease and neurological disorder relating to neurodegeneration. Anotherembodiment is when the reperfusion injury is a vascular stroke. Yetanother embodiment is when the peripheral neuropathy is caused byGuillain-Barre syndrome. Still another embodiment is when thedemyelinating disease and neurological disorder relates toneurodegeneration. Another embodiment is when the reperfusion injury isa vascular stroke. Still another preferred embodiment is when thedemyelinating disease is multiple sclerosis. Another embodiment is whenthe neurological disorder relating to neurodegeneration is selected fromthe group consisting of Alzheimer's Disease, Parkinson's Disease, andamyotrophic lateral sclerosis.

Another embodiment is a method of treating, preventing or inhibiting acardiovascular disease in an animal, such as angina pectoris, myocardialinfarction, cardiovascular ischemia, and cardiovascular tissue damagerelated to PARP activation, by administering to said animal an effectiveamount of the compounds of the present invention.

The present invention also contemplates the use of a compound thepresent invention for inhibiting PARP activity, for treating, preventingor inhibiting tissue damage resulting from cell damage or death due tonecrosis or apoptosis, for treating, preventing or inhibiting aneurological disorder in an animal.

In another embodiment, the neurological disorder is selected from thegroup consisting of peripheral neuropathy caused by physical injury ordisease state, traumatic brain injury, physical damage to the spinalcord, stroke associated with brain damage, focal ischemia, globalischemia, reperfusion injury, demyelinating disease and neurologicaldisorder relating to neurodegeneration.

Another embodiment is when the reperfusion injury is a vascular stroke.Yet another embodiment is when the peripheral neuropathy is caused byGuillain-Barre syndrome. Still another embodiment is when thedemyelinating disease is multiple sclerosis. Another embodiment is whenthe neurological disorder relating to neurodegeneration is selected fromthe group consisting of Alzheimer's Disease, Parkinson's Disease, andamyotrophic lateral sclerosis.

The present invention also contemplates the use of a compound of thepresent invention in the preparation of a medicament for the treatmentof any of the diseases and disorders in an animal described herein.

In another embodiment, the disease or disorder is a neurologicaldisorder.

In another embodiment, the neurological disorder is selected from thegroup consisting of peripheral neuropathy caused by physical injury ordisease state, traumatic brain injury, physical damage to the spinalcord, stroke associated with brain damage, focal ischemia, globalischemia, reperfusion injury, demyelinating disease and neurologicaldisorder relating to neurodegeneration. Another embodiment is when thereperfusion injury is a vascular stroke. Yet another embodiment is whenthe peripheral neuropathy is caused by Guillain-Barre syndrome.

Still another embodiment is when the demyelinating disease is multiplesclerosis. Another embodiment is when the neurological disorder relatingto neurodegeneration is selected from the group consisting ofAlzheimer's Disease, Parkinson's Disease, and amyotrophic lateralsclerosis.

In another embodiment of the present invention, a person diagnosed withacute retinal ischemia or acute vascular stroke is immediatelyadministered parenterally, either by intermittent or continuousintravenous administration, a compound of the present invention eitheras a single dose or a series of divided doses of the compound. Afterthis initial treatment, and depending on the person's presentingneurological symptoms, the person optionally may receive the same or adifferent compound of the invention in the form of another parenteraldose. The compound of the invention can be administered by intermittentor continuous administration via implantation of a biocompatible,biodegradable polymeric matrix delivery system containing the compound,or via a subdural pump inserted to administer the compound directly tothe infarct area of the brain.

In another embodiment, the present invention provides methods to extendthe lifespan and proliferative capacity of cells, such as, for example,in using the compounds of the invention as general mediators in thegeneration of oxidants, proinflammatory mediators and/or cytokines,and/or general mediators of leukocyte infiltration, calcium ionoverload, phospholipid peroxidaion, impaired nitric oxide metabolismand/or reduced ATP production

Further still, the methods of the invention can be used to treat cancerand to radiosensitize tumor cells. The term “cancer” is interpretedbroadly. The compounds of the present invention can be “anti-canceragents”, which term also encompasses “anti-tumor cell growth agents” and“anti-neoplastic agents”. For example, the methods of the invention areuseful for treating cancers and radiosensitizing tumor ceils in cancerssuch as ACTH-producing tumors, acute lymphocytic leukemia, acutenonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer,brain cancer, breast cancer, cervical cancer, chronic lymphocyticleukemia, chronic myelocytic leukemia, colorectal cancer, cutaneousT-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma,gallbladder cancer, hairy cell leukemia, head & neck cancer, Hodgkin'slymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer(small and/or non-small cell), malignant peritoneal effusion, malignantpleural effusion, melanoma, mesothelioma, multiple myeloma,neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer,ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penilecancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cellcarcinomas, stomach cancer, testicular cancer, thyroid cancer,trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of thevulva and Wilm's tumor.

The methods of the present invention can also treat cancer in a mammalwith an effective amount of temozolimide and a compound of the presentinvention. The cancer can be melanoma, lymphoma, and glioblastomamultiforme.

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of electromagnetic radiation. Severalmechanisms for the mode of action of radiosensitizers have beensuggested in the literature including: hypoxic cell radiosensitizers(e.g., 2-nitroimidazole compounds, and benzotriazine dioxide compounds)promote the reoxygenation of hypoxic tissue and/or catalyze thegeneration of damaging oxygen radicals; non-hypoxic cellradiosensitizers (e.g., halogenated pyrimidines) can be analogs of DNAbases and preferentially incorporate into the DNA of cancer cells andthereby promote the radiation-induced breaking of DNA molecules and/orprevent the normal DNA repair mechanisms; and various other potentialmechanisms of action have been hypothesized for radiosensitizers in thetreatment of disease.

Many cancer treatment protocols currently employ radiosensitizersactivated by the electromagnetic radiation of x-rays. Examples of x-rayactivated radiosensitizers include, but are not limited to, thefollowing: metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine(FudR), hydroxyurea, cisplatin, and therapeutically effective analogsand derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives,NPe6, tin etioporphyrin SnET2, pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

Radiosensitizers may be administered in conjunction with atherapeutically effective amount of one or more other compounds,including but not limited to: compounds which promote the incorporationof radiosensitizers to the target cells; compounds which control theflow of therapeutics, nutrients, and/or oxygen to the target cells;chemotherapeutic agents which act on the tumor with or withoutadditional radiation; or other therapeutically effective compounds fortreating cancer or other disease. Examples of additional therapeuticagents that may he used in conjunction with radiosensitizers include,but are not limited to: 5-fluorouracil, leucovorin,5′-amino-5′deoxythymidine, oxygen, carbogen, red cell transfusions,perfluorocarbons (e.g., Fluosol-DA), 2,3-DPG, BWI2C, calcium channelblockers, pentoxyfylline, antiangiogenesis compounds, hydralazine, andLBSO. Examples of chemotherapeutic agents that may be used inconjunction with radiosensitizers include, but are not limited to:adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2,irinotecan, paclitaxel, topotecan, and therapeutically effective analogsand derivatives of the same.

The present invention provides means to treat chemotherapy-inducedperipheral neuropathy. According to an aspect of the invention, thecompounds of the present invention are administered prior to, ortogether with, the administration of at least one chemotherapy agent toprevent the development of neuropathy symptoms or to mitigate theseverity of such symptoms. According to a further aspect, the compoundsof the present invention are administered after the administration of atleast one chemotherapeutic agent to cure a patient of the symptoms ofneuropathy or to mitigate the severity of such symptoms. In anotheraspect, the present invention provides a method to retard, delay, orarrest the growth of tumor cells in a mammal, comprising theadministration of a chemotherapeutic agent, and further comprising theadministration of a compound of Group I in an amount sufficient topotentiate the antitumor activity of said chemotherapeutic agent.

In another embodiment compounds of the invention act as PARP inhibitorsto treat or prevent cancers by chemopotentiating the cytotoxic effectsof other chemotherapeutic agents.

The present invention provides compounds of Group I, derivativesthereof, and compositions containing these compounds to treat, preventand/or ameliorate the effects of cancers by potentiating the cytotoxiceffects of ionizing radiation on tumor cells.

In another embodiment the present invention provides compounds describedherein, derivatives thereof, and compositions containing these compoundsto treat, prevent, and/or ameliorate the effects of cancers bypotentiating the cytotoxic effects of ehemotherapeutic agents on tumorcells.

In another embodiment the methods of the invention can be used to treatcancer and to chemosensitize tumor cells. The term “cancer,” as usedherein, is defined broadly. The compounds of the present invention canpotentiate the effects of “anti-cancer agents,” which term alsoencompasses “anti-tumor cell growth agents,” “chemotherapeutic agents,”“cytostatic agents,” “cytotoxic agents,” and “anti-neoplastic agents”.

In one embodiment methods of the invention are useful for treatingcancers and radiosensitizing or chemosensitizing tumor cells in cancerssuch as ACTH-producing tumors, acute lymphocytic leukemia, acutenonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer,brain cancer, breast cancer, cervical cancer, chronic lymphocyticleukemia, chronic myelocytic leukemia, colorectal cancer, cutaneousT-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma,gallbladder cancer, hairy cell leukemia, head & neck cancer, Hodgkin'slymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer(small and/or non-small cell), malignant peritoneal effusion,malignant-pleural effusion, melanoma, mesothelioma, multiple myeloma,neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer,ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penilecancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cellcarcinomas, stomach cancer, testicular cancer, thyroid cancer,trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of thevulva and Wilm's tumor.

The present invention provides a chemosensitization method for treatingtumor and/or cancer cells comprising contacting said cancer cells with adiazabenzo[de]anthracen-3-one compound of Group I and further contactingsaid cancer cells with an anticancer agent.

Specific embodiments of the present invention include thediazabenzo[de]anthracen-3-one compounds shown in Group I and neutraland/or salt forms thereof, as well as enantiomer and racemic mixturesthereof, where appropriate.

The compounds of the present invention may possess one or moreasymmetric center(s) and thus can be produced as mixtures (racemic andnon-racemic) of stereoisomers, or as individual enantiomers ordiastereomers. The individual stereoisomers may be obtained by using anoptically active staring material, by resolving a racemic or non-racemicmixture of an intermediate at some appropriate stage of the synthesis,or by resolution of the compound of Group I. It is understood that theindividual stereoisomers as well as mixtures (racemic and non-racemic)of stereoisomers are encompassed by the scope of the present invention.

The compounds of the invention are useful in a free base form, in theform of pharmaceutically acceptable salts, pharmaceutically acceptablehydrates, pharmaceutically acceptable esters, pharmaceuticallyacceptable solvates, pharmaceutically acceptable prodrugs,pharmaceutically acceptable metabolites, and in the form ofpharmaceutically acceptable stereoisomers. These forms are all withinthe scope of the invention.

“Pharmaceutically acceptable salt”, “hydrate”, “ester” or “solvate”refers to a salt hydrate, ester, or solvate of the inventive compoundswhich possesses the desired pharmacological activity and which isneither biologically nor otherwise undesirable. Organic acids can beused to produce salts, hydrates, esters, or solvates such as acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate,p-toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate,butyrate, citrate, camphorate, camphorsulfonate,cyclopentane-propionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate heptanoate,hexanoate, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate.Inorganic acids can be used to produce salts, hydrates, esters, orsolvates such as hydrochloride, hydrobromide, hydroiodide, andthiocyanate.

Examples of suitable base salts, hydrates, esters, or solvates includehydroxides, carbonates, and bicarbonates of ammonia, alkali metal saltssuch as sodium, lithium and potassium salts, alkaline earth metal saltssuch as calcium and magnesium salts, aluminum salts, and zinc salts.

Salts, hydrates, esters, or solvates may also be formed with organicbases. Organic bases suitable for the formation of pharmaceuticallyacceptable base addition salts, hydrates, esters, or solvates of thecompounds of the present invention include those that are non-toxic andstrong enough to form such salts, hydrates, esters, or solvates. Forpurposes of illustration, the class of such organic bases may includemono-, di- and trialkylamines, such as methylamine, dimethylamine,triethylamine and dicyclohexylamine; mono-, di- ortrihydroxyalkylamines, such as mono-, di, and triethanolamine; aminoacids, such as arginine and lysine; guanidine; N-methyl-glucosamine;N-methyl-glucamine; L-glutamine; N-methyl-piperazine; morpholine;ethylenediamine; N-benzyl-phenethylamine;(trihydroxy-methyl)aminoethane; and the life. See, for example,“Pharmaceutical Salts,” J. Pharm. Sci., 66:1, 1-19 (1977). Accordingly,basic nitrogen-containing groups can be quaternized with agentsincluding: lower alkyl halides such as methyl, ethyl, propyl, and butylchlorides, bromides and iodides; dialkyl sulfates such as dimethyl,diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides; andaralkyl halides such as benzyl and phenethyl bromides.

The acid addition salts, hydrates, esters, or solvates of the basiccompounds may be prepared either by dissolving the free base of acompound of the present invention in an aqueous or an aqueous alcoholsolution or other suitable solvent containing the appropriate acid orbase, and isolating the salt by evaporating the solution. Alternatively,the free base of a compound of the present invention can he reacted withan acid, as well as reacting a compound of the present invention havingan acid group thereon with a base, such that the reactions are in anorganic solvent, in which case the salt separates directly or can beobtained by concentrating the solution.

“Pharmaceutically acceptable prodrug” refers to a derivative of theinventive compounds which undergoes biotransformation prior toexhibiting its pharmacological effect(s). The prodrug is formulated withthe objective(s) of improved chemical stability, improved patientacceptance and compliance, improved bioavailability, prolonged durationof action, improved organ selectivity, improved formulation (e.g.,increased hydrosolubility), and/or decreased side effects (e.g.,toxicity). The prodrug can be readily prepared from the inventivecompounds using methods known in the art, such as those described byBurgers Medicinal Chemistry and Drug Chemistry, Fifth Ed, Vol. 1, pp.172-178, 949-982 (1995). For example, the inventive compounds can betransformed into prodrugs by converting one or more of the hydroxy orcarboxy groups into esters.

“Pharmaceutically acceptable metabolite” refers to drugs that haveundergone a metabolic transformation. After entry into the body, mostdrugs are substrates for chemical reactions that may change theirphysical properties and biologic effects. These metabolic conversions,which usually affect the polarity of the compound, alter the way inwhich drugs are distributed in and excreted from the body. However, insome cases, metabolism of a drug is required for therapeutic effect. Forexample, anticancer drugs of the antimetabolite class must be convertedto their active forms after they have been transported into a cancercell. Since most drugs undergo metabolic transformation of some kind,the biochemical reactions that play a role in drug metabolism may benumerous and diverse. The main site of drug metabolism is the liver,although other tissues may also participate.

Pharmaceutical Compositions of the Invention

The present invention also relates to a pharmaceutical compositioncomprising (i) a therapeutically effective amount of a compound of adiazabenzo[de]anthracen-3-one derivative and (ii) a pharmaceuticallyacceptable carrier.

The above discussion relating to the preferred embodiments' utility andadministration of the compounds of the present invention also applies tothe pharmaceutical composition of the present invention.

The term “pharmaceutically acceptable carrier” as used herein refers toany carrier, diluent, excipient, suspending agent, lubricating agent,adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent,preservative, surfactant, colorant, flavorant, or sweetener.

For these purposes, the composition of the invention may be administeredorally, parenterally, by inhalation spray, adsorption, absorption,topically, rectally, nasally, bucally, vaginally, intraventricularly,via an implanted reservoir in dosage formulations containingconventional non-toxic pharmaceutically-acceptable carriers, or by anyother convenient dosage form. The term parenteral as used hereinincludes subcutaneous, intravenous, intramuscular, intraperitoneal,intrathecal intraventricular, intrasternal, and intracranial injectionor infusion techniques.

When administered parenterally, the composition will normally be in aunit dosage, sterile injectable form (solution, suspension or emulsion)which is preferably isotonic with the blood of the recipient with apharmaceutically acceptable carrier. Examples of such sterile injectableforms are sterile injectable aqueous or oleaginous suspensions. Thesesuspensions may be formulated according to techniques known in the artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable forms may also be sterile injectable solutions orsuspensions in non-toxic parenterally-acceptable diluents or solvents,for example, as solutions in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, saline, Ringer'ssolution, dextrose solution, isotonic sodium chloride solution, andHanks' solution. In addition, sterile, fixed oils are conventionallyemployed as solvents or suspending mediums. For this purpose, any blandfixed oil may be employed including synthetic mono- or di-glycerides,corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyloleate, isopropyl myristate, and oleic acid and its glyceridederivatives, including olive oil and castor oil, especially in theirpolyoxyethylated versions, are useful in the preparation of injectables.These oil solutions or suspensions may also contain long-chain alcoholdiluents or dispersants.

Sterile saline is a preferred carrier, and the compounds are oftensufficiently water soluble to be made up as a solution for allforeseeable needs. The carrier may contain minor amounts of additives,such as substances that enhance solubility, isotonicity, and chemicalstability, e.g., anti-oxidants, buffers and preservatives.

Formulations suitable for nasal or buccal administration (such asself-propelling powder dispensing formulations) may comprise about 0.1%to about 5% w/w, for example 1% w/w of active ingredient. Theformulations for human medical use of the present invention comprise anactive ingredient in association with a pharmaceutically acceptablecarrier therefore and optionally other therapeutic ingredient(s).

When administered orally, the composition will usually be formulatedinto unit dosage forms such as tablets, cachets, powder, granules,beads, ehewable lozenges, capsules, liquids, aqueous suspensions orsolutions, or similar dosage forms, using conventional equipment andtechniques known in the art. Such formulations typically include asolid, semisolid, or liquid carrier. Exemplary carriers include lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, mineral oil, cocoa butter, oil of theobroma, alginates,tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitanmonolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,magnesium stearate, and the like.

The composition of the invention is preferably administered as a capsuleor tablet containing a single or divided dose of the compound of GroupI. Preferably, the composition is administered as a sterile solution,suspension, or emulsion, in a single or divided dose. Tablets maycontain carriers such as lactose and corn starch, and/or lubricatingagents such as magnesium stearate. Capsules may contain diluentsincluding lactose and dried corn starch.

A tablet may be made by compressing or molding the active ingredientoptionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, surfaceactive, or dispersing agent Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active ingredient and asuitable carrier moistened with an inert liquid diluent.

The compounds of this invention may also be administered rectally in theform of suppositories. These compositions can be prepared by mixing thedrug with a suitable non-irritating excipient which is solid at roomtemperature, but liquid at rectal temperature, and, therefore, will meltin the rectum to release the drug. Such materials include cocoa butter,beeswax, and polyethylene glycols,

Compositions and methods of the invention also may utilize controlledrelease technology. Thus, for example, the inventive compounds may beincorporated into a hydrophobic polymer matrix for controlled releaseover a period of days. The composition of the invention may then bemolded into a solid implant, or externally applied patch, suitable forproviding efficacious concentrations of the PARP inhibitors over aprolonged period of time without the need for frequent re-dosing. Suchcontrolled release films are well known to the art. Particularlypreferred are transdermal delivery systems. Other examples of polymerscommonly employed for this purpose that may be used in the presentinvention include nondegradable ethylene-vinyl acetate copolymer adegradable lactic acid-glycolic add copolymers which may be usedexternally or internally. Certain, hydrogels such aspoly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful,but for shorter release cycles than the other polymer release systems,such as those mentioned above.

In a preferred embodiment, the carrier is a solid biodegradable polymeror mixture of biodegradable polymers with appropriate time releasecharacteristics and release kinetics. The composition of the inventionmay then be molded into a solid implant suitable for providingefficacious concentrations of the compounds of the invention over aprolonged period of time without the need for frequent re-dosing. Thecomposition of the present invention can be incorporated into thebiodegradable polymer or polymer mixture in any suitable manner known toone of ordinary skill in the art and may form a homogeneous matrix withthe biodegradable polymer, or may be encapsulated in some way within thepolymer, or may be molded into a solid implant

In one embodiment, the biodegradable polymer or polymer mixture is usedto form a soft “depot” containing the pharmaceutical composition of thepresent invention that can be administered as a flowable liquid, forexample, by injection, but which remains sufficiently viscous tomaintain the pharmaceutical composition within the localized area aroundthe injection site. The degradation time of the depot so formed can bevaried from several days to a few years, depending upon the polymerselected and its molecular weight. By using a polymer composition ininjectable form, even the need to make an incision may be eliminated. Inany event, a flexible or flowable delivery “depot” will adjust to theshape of the space it occupies with the body with a minimum of trauma tosurrounding tissues. The pharmaceutical composition of the presentinvention is used in amounts that are therapeutically effective, and maydepend upon the desired release profile, the concentration of thepharmaceutical composition required for the sensitizing effect, and thelength of time that the pharmaceutical composition has to be releasedfor treatment.

The compounds of the invention are used in the composition in amountsthat are therapeutically effective. The compositions may be sterilizedand/or contain adjuvants, such as preserving, stabilizing, welling, oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In addition, they may also contain othertherapeutically valuable substances, such, as, without limitation, thespecific chemotherapeutic agents recited herein. The compositions areprepared according to conventional mixing, granulating, or coatingmethods, and contain about 0.1 to 75% by weight, preferably about 1 to50% by weight, of the compound of the invention.

To be effective therapeutically as central nervous system targets, thecompounds of the present invention should readily penetrate theblood-brain barrier when peripherally administered. Compounds whichcannot penetrate the blood-brain barrier can he effectively administeredby an intraventricular route or other appropriate delivery systemsuitable for administration to the brain.

For medical use, the amount required of the active ingredient to achievea therapeutic effect will vary with the particular compound, the routeof administration, the mammal under treatment, and the particulardisorder or disease being treated. A suitable systematic dose of acompound of the present invention or a pharmacologically acceptable saltthereof for a mammal suffering from, or likely to suffer from, any ofcondition as described hereinbefore is in the range of about 0.1 mg/kgto about 1.00 mg/kg of the active ingredient compound, the mostpreferred dosage being about 1 to about 10 mg/kg.

It is understood, however, that a specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, rate of excretion, drug combination,and the severity of the particular disease being treated and form ofadministration.

It is understood that the ordinarily skilled physician or veterinarianwill readily determine and prescribe the effective amount of thecompound for prophylactic or therapeutic treatment of the condition forwhich treatment is administered. In so proceeding, the physician orveterinarian can, for example, employ an intravenous bolus followed byan intravenous infusion and repeated administrations, parenterally ororally, as considered appropriate. While it is possible for an activeingredient to be administered alone, it is preferable to present it as aformulation.

When preparing dosage form incorporating the compositions of theinvention, the compounds may also be blended with conventionalexcipients such as binders, including gelatin, pregelatinized starch,and the like; lubricants, such as hydrogenated vegetable oil, stearicacid, and the like; diluents, such as lactose, mannose, and sucrose;disintegrants, such as carboxymethylcellulose and sodium starchglyeolate; suspending agents, such as povidone, polyvinyl alcohol, andthe like; absorbants, such as silicon dioxide; preservatives, such asmethylparaben, propylparaben, and sodium benzoate; surfactants, such assodium lauryl sulfate, polysorbate 80, and the like; colorants such asF.D. & C, dyes and lakes; flavorants; and sweeteners.

The present invention relates to the use of a compound of Group I in thepreparation of a medicament for the treatment of any disease or disorderin an animal described herein. In an embodiment, the compounds of thepresent invention are used to treat cancer. In a preferred embodiment,the compounds of the present invention are used to potentiate thecytotoxic effects of ionizing radiation. In such an embodiment, thecompounds of the present invention act as a radiosensitizer. In analternative preferred embodiment, the compounds of the present inventionare used to potentiate the cytotoxic effects of chemotherapeutic agents.In such an embodiment, the compounds of the present invention act as achemosensitizer.

Any pharmacologically-acceptable chemotherapeutic agent that acts bydamaging DNA is suitable as the chemotherapeutic agent of the presentinvention. In particular, the present invention contemplates the use ofa chemotherapeutically effective amount of at least one chemotherapeuticagent including, but not limited to: temozolomide, adriamycin,camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel,doxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan,paclitaxel, topotecan, therapeutically effective analogs and derivativesof the same, and mixtures thereof. According to a preferred aspect, thechemotherapeutic agent is temozolomide.

The disclosure contained herein demonstrates the usefulness of thecompounds and compositions of the present invention in treating and/orpreventing cancer, such as by radiosensitizing and/or chemosensitizingtumor and/or cancer cells to chemotherapeutic agents.

EXAMPLES

The diazabenzo[de]anthracen -3-one compounds of the present inventioncan be synthesized using the starting materials and methods disclosed inU.S. 60/644,584, which is incorporated herein by reference in itsentirety for all purposes.

Some of the PARP inhibitors used in the inventive methods andpharmaceutical compositions can be readily prepared by standardtechniques of organic chemistry, utilizing the general syntheticpathways and examples depicted in publications. See, e.g., Wu et al,“The Protective Effect of GPI 18078, a Novel Water Soluble Poly(ADP-Ribose) Polymerase Inhibitor in Myocardial Ischemia-ReprefusionInjury, Experimental Biology,” FASEB, Apr. 11-15 (2003); Wu et al.,“Myocardial Protection and Anti-Inflammatory Effect of GPI 15427, aNovel Wafer Soluble Poly (ADP-Ribose) Polymerase Inhibitor: Comparisonwith GPI 6150, Experimental Biology,” FASEB, Apr. 11-15 (2003); Kalishet al., “Design, Synthesis and SAR of PARP-1 Inhibitors, ISMC Meeting,Barcelona,” Sep. 4, 2002; Xu et al., “Design and Synthesis of NovelPotent Poly (ADP-Ribose) Polymerase (PARP) Inhibitors, 224^(th) ACSNational Meeting,” Boston, Aug. 18-23 (2002); Williams et al.,“Intravenous Delivery of GPI 15427/C and GPI 16539/C, PotentWater-Soluble PARP Inhibitors, Reduces Infarct Volume FollowingPermanent and Transient Focal Cerebral Ischemia, Soceity forNeuroscience,” Orlando Fl., October (2002); Tentori L, et al., “Systemicadministration of the PARP-1 inhibitor GPI 15427 increases theanti-tumor activity of temozolomide against metastatic melanoma,”Medical Science Monitor, Vol. 9, supplement 1, 34 (2003); Tentori etal., “Poly(ADP-Ribose) Polymerase Inhibitor to Increase TemozolomideEfficacy Against Melanoma, Glioma and Lymphoma at the CNS Site,” AACRposter, April (2003); Suto et al., “Dihydroiso-quinolinones: The Designand Synthesis of a New Series of Potent Inhibitors of Poly(ADP-ribose)Polymerase,” Anticancer Drug Des., 6:107-17 (1991): and U.S. Pat. Nos.6,348,475, 6,545,011, RE36,397, 6,380,211, 6,235,748, 6,121,278,6,197,785, 6,380,193, 6,346,536, 6,514,983, 6,306,889, 6,387,902,6,201,020, and 6,291,425, and U.S. patent application Ser. No.10/853,714, the entire contents of which patents, patent application andpublications are herein Incorporated by reference, as though set forthherein in full.

The compounds of this invention can be prepared in a conventional manneras illustrated below in Schemes 1. Starting derivatives are known in thechemistry literature and accessible by processes known to one skilled Inthe art.

General Procedure A Preparation of7-bromomethyl-9-oxoxanthene-1-carboxylic acid methyl ester

Brominating agents including N-bromosuccinimide, bromine and complexedbromine such as pyridinium bromide can be used to convert7-methyl-9-oxoxanthene-1 -carboxylic acid methyl ester, 1, to7-bromomethyl-9-oxoxanthene-1-carboxylic acid methyl ester, 2. Suitablesolvents include, but are not limited to, chlorinated hydrocarbons,polar aprotic solvents, as well as various ethers. Temperatures aregenerally between 0 and 100° C., with a range of 50-70° C. beingpreferred.

Example 1

To a stirred solution of compound 5 (400 g, 1.49 mol) in refluxingcarbon tetrachloride (10 L) containing benzoyl peroxide (10 g, 0.041mol) was added NBS (292 g, 1.64 moi) in several portions over 45minutes. The resulting mixture was refluxed for 12 hours and then cooledto room temperature overnight. The precipitated was filtered off and thecake was washed with water (1.2 L) thoroughly and dried to give 322 g ofcompound 6 as a white solid (62%).

Example 2

To a solution of compound 1 (1.97 g, 7.3 mmol, 1.00 eq) in carbontetrachloride (400 mL) was added N-bromosuccinimide (1.44 g, 8.1 mmol,1.10 eq) and a catalytic amount of benzoyl peroxide (45 mg, 0.2 mmol,catalytic). The reaction mixture was heated to reflux for 6 hours andthen cooled to room temperature. The resulting white precipitate wasisolated via vacuum filtration. Residual solvents were removed and thefilter cake was twice recrystallized from ethyl acetate and hexanes toyield a white solid, 2. (1.15 g, 45%). ¹H NMR (400 MHz, CDCl₃) 8.27 (d,J=2.5 Hz, 1H), 7.72-7.80 (m, 2H), 7.57 (dd, J=8.5 and 1.1 Hz, 1H), 7.48(d, J=8.5 Hz, 1H), 7.33 (dd, J=7.0 and 1.1 Hz, 1H), 4.57 (s, 2H), 4.00(s. 3H). ¹³C NMR (400 MHz, CDCl₃) 31.97, 53.06, 118.63, 118.71, 119.63,121.56, 122.81, 126.83, 134.15, 134.18, 134.50, 135.95, 155.31, 155.87,169.72, 175.48.

General Procedure B Preparation of Substituted9-oxoxanthene-1-carboxylic acid methyl esters

The primary bromide in compound 2 can be readily displaced bynucleophiles which include primary and secondary amines and ispreferably done so in the presence of a non-reactive basic species, suchas potassium carbonate. Suitable solvents for this transformation arepolar and aprotic such as dimethylformamide or acetonitrile, but thereaction may also be performed in other media. The temperature may rangefrom. 0-100° C. with 50-80° C. being preferred.

Example 1

To a solution of compound 2 (3.47 g, 10.0 mmol, 1.00 eq) indimethylformamide (100 mL) is added potassium carbonate (13.82 g, 100.0mmol, 10.00 eq) and a secondary amine (10 mmol 1 eq). The reactionmixture is heated to 70° C. for 6 hours and then cooled to roomtemperature. Water (100 mL) is added to the reaction mixture, followedby ethyl acetate (200 mL). The organic layer is collected, washed withwater followed by brine and then dried over sodium or magnesium sulfate.The solvents are removed in vacuo and the residue is purified by columnchromatography using ethyl acetate and hexanes as an eluent to giveproduct 3 in 50-90% yields.

Example 2

To a solution of compound 2 (1.53 g, 4.4 mmol, 1.00 eq) in acetonitrile(50 mL) was added potassium carbonate (1.2 g, 8.7 mmol, 2.00 eq) and1-methypiperazine (0.51 mL, 4,6 mmol. 1.05 eq). The reaction mixture wasthen heated to reflux overnight. After cooling to room temperature, thesolids were removed by filtration and the organics were evaporated to anoily residue. This material was dissolved in ethyl acetate (150 ml) andextracted with 1 N HCl (150 ml). The organic layer was discarded and thepH of the aqueous layer was adjusted to greater than 9 with 6 N sodiumhydroxide. The product was then extracted with two portions of ethylacetate (100 ml) which were subsequently combined, washed successivelywith water and brine and then dried over magnesium sulfate. All solventswere removed in vacuo to afford 3a as a white solid. (0.95 g, 59%). ¹HNMR (400 MHz, CDCl₃) of 3a: 8.18 (d, J=2.5 Hz, 1H), 7.71-7.75 (m, 2H),7.56 (dd, J=8.5 and 1.1 Hz, 1H), 7.45 (d, J=8.5 Hz, 1H), 7.32 (dd, J=7.0and 1.1 Hz, 1H), 4.04 (s, 3H), 3.58 (s, 2H), 2.45 (br, 8H), 2.27 (s,3H).

General Procedure C Preparation of10-aminomethyl-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one Derivatives

Cyclization of the ketone and methyl ester in compound 3 to formbenzopyrano[4,3,2-de]phthalazine rings can be performed with hydrazineto afford 10-aminomethyl-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-onederivatives, 4, in high yield. Ethanol is the preferred solvent, but thereaction is not exclusively limited to this media. Temperatures canrange from 0-20° C., with 70-90° C. being most desirable.

Example1

To a solution of 3 (5 mmol) in absolute ethanol (10 mL) is addedanhydrous hydrazine in ethanol (1 mL) drop wise at room temperature.After the addition is complete, the solution is heated to refluxovernight. Once cooled to room temperature, ice-cold water (100 mL) isadded and white solid is precipitated. The solid is collected by vacuumfiltration, washed successively with water and ethanol and then dried invacuo to afford 4 as a white solid (40-85% yield).

Example 2

7-(1,4-Dioxa-8-aza-spiro[4.5]dec-8-ylmethyl)-9-oxo-9H-xanthene-1-carboxylicacid methyl ester, 3a (1.6 g, 3.91 mmol, 1 eq), in ethanol (55 ml) washeated to 80° C. and stirred until all material was in solution. To thishydrazine monohydrate (20 ml, large excess) was added dropwise over tenminutes. The reaction mixture was heated to reflux overnight duringwhich time a heavy white precipitate formed. The solution is allowed tocool to room temperature and product is isolated by vacuum filtration.Washing with successive small portions of water, ethanol and pentane andthen subsequent drying in vacuo affords 4n in high yield (1.4 g, 92%).¹H-NMR (DMSO-d₆, 300 MHz): 1.62 (t, J=5.0, 4H), 2.40-2.50 (m, 4H), 3.55(s, 2H), 3.85 (s, 4H), 7.34 (d, J=9.4 Hz, 1H), 7.47 (d, J=7.5 Hz, 1H),7.67-7.70 (m, 1H), 7.86-7.92 (m, 2H), 7.98 (s, 1H), 12.62 (s, 1H).

Compound 4a:10-(4-Isopropyl-piperazin-1-ylmethyl)2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and 1-isopropyl-piperazine according to generalprocedures B and C. Purification of the product by crystallization fromethanol gave 4a as a white solid. MS (ES+): 377. ¹H-NMR (CDCl₃, 300MHz): 0.90-1.00 (m, 6H), 2.25-2.50 (m, 8H), 2.55-2.60 (m, 1H), 3.34 (s,2H), 7.35 (d, 1H), 7.46 (d, 1H), 7.68 (dd, 1H), 7.80-7.95 (m, 2H),7.95-8.05 (m, 1H), 12.63 (s. 1H). Anal. Calcd. for C₂₂H₂₄N₄O₂: C, 70.19;H, 6.43; N, 14.88. Found: C, 70.09; H, 6.51; N, 14.77.

Compound 4b:10-[4-(2-Methoxy-ethyl)-piperazin-1-ylmethyl]-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and 1-(2-methoxyethyl)piperazine according togeneral procedures B and C. Purification of the product bycrystallization from ethanol gave 4b as a white solid. MS (ES+): 393;¹H-NMR (DMSO-d₆, 300 MHz): 2.30-2.49 (m, 10H), 3.22 (s, 3H), 3.30-3.45(m, 2H), 3.50 (m, 2H), 7.30-7.35 (m, 1H), 7.40-7.48 (m, 1H), 7.65-7.70(m, 1H), 7.85-7.95 (m, 2H), 7.95-8.05 (m, 1H), 12.63(s, 1H). Anal.Calcd. for C₂₂H₂₄N₄O₃: C, 67.33; H, 6.16; N, 14.28. Found: C, 67.35; H,6.16; N, 14.45.

Compound 4c:10-(4-Pyrimidin-2-yl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and 1-(2-pyrimidyl)piperazine according togeneral procedures B and C. Purification of the product bycrystallization from ethanol gave 4c as a white solid. MS (ES+): 413.¹H-NMR (DMSO-d₆, 300 MHz): 3.28-3.34 (m, 4H), 3.60 (s, 2H) 3.70-3.78 (m,4H), 6.60-6.62 (m, 1H), 7.38-7.40 (m, 1H), 7.50-7.60 (m, 1H), 7.70-7.74(m, 1H), 7.80-7.95 (m, 2H), 8.05-8.10 (m, 1H), 8.30-8.40 (m, 2H) 12.64(s, 1H). Anal. Calcd. for C₂₃H₂₀N₆O₂: C, 66.98; H, 4.89; N, 20.38.Found: C, 67.03; H, 4.88; N, 20.15.

Compound 4d:10-(3-Oxo-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and piperasin-2-one according to generalprocedures B and C. Purification of the product by crystallization fromethanol gave 4d as a white solid. MS (ES+): 349; ¹H-NMR (DMSO-d₆, 300MHz): 2.58-2.62 (m, 2H), 2.94 (s, 2H), 3.16-3.20 (m, 2H), 3.63 (s, 2H),7.30-7.35 (m, 1H), 7.40-7.48 (m, 1H), 7.65-7.70 (m, 1H), 7.85-7.95 (m,2H), 7.95-8.05 (m, 1H).

Compound 4e:10-[4-(2-Pyrrolidin-1-yl-ethyl)-piperazin-1-ylmethyl]-2H-7-oxa-1,2,diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and 1-(2-Pyrrolidin-1-yl-ethyl)-piperazineaccording to general procedures B and C. Purification of the product bycrystallization from ethanol gave 4e as a white solid. ¹H-NMR (DMSO-d₆,300 MHz): 1.64 (m, 4H), 2.30-2.55 (m, 16H), 3.52 (s, 2H), 7.30-7.40 (m,1H), 7.45-7.50 (m, 1H), 7.70-7.75 (m, 1H), 7.80-7.90 (m,2H), 8.00-8.05(m, 1H). Anal. Calcd. for C₂₅H₂₉N₅O₂.(0.7 H₂0): C, 67.61; H, 6.90; N,15.77; Found: C, 67.25; H, 6.81; N, 15.67.

Compound 4f:10-[4-(3-Dimethylamino-propyl)-piperazin-1-ylmethyl]-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and Dimethyl-(3-piperazin-1-yl-propyl)-amineaccording to general procedures B and C. Purification of the product bycrystallization from ethanol gave 4f as a white solid. ¹H-NMR (DMSO-d₆,300MHz): 1.45-1.55 (m, 2H), 2.09 (s, 6H), 2.10-2.40 (m, 12H), 3.52 (s,2H), 7.30-7.40 9m, 1H), 7.40-7.50 (m, 1H), 7.70-7.75 (m, 1H), 7.80-7.95(m, 2H), 8.01 (s, 1H).

Compound 4g:10-{[(2-Diethylamino-ethyl-amino)-ethyl-amino]-methyl}-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and N,N-Diethyl-N′-ethyl-ethane-1,2-diamineaccording to general procedures B and C. Purification of the product bycrystallization from ethanol gave 4g as a white solid. ¹H-NMR (DMSO-d₆,300 MHz): 0.90 (t, J=7.2 Hz, 6H), 1.00 (t, J=6.8 Hz, 3H), 2.41 (dd,J=14.3 and 7.2 Hz, 4H), 2.45-2.55 (m, 6H), 3.62 (s, 2H), 7.35 (d, J=8.6Hz, 1H), 7.49 (dd, J=8.3 and 2,3 Hz, 1H), 7.69 (dd, J=7.1 and 2.4 Hz,1H), 7.86-7.93 (m, 2H), 8.03 (d, J=2.3 Hz, 1H)

Compound 4h:10-{[(2-Diethylamino-ethyl)-methyl-amino]-methyl}-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and N,N-Diethyl-N′-methyl-ethane-1,2-diamineaccording to general procedures B and C. Purification of the product bycrystallization from ethanol gave 4h as a white solid. ¹H-NMR (DMSO-d₆,300 MHz): 12.63 (s, 1H), 8.00 (d, J=1.9 Hz, 1H), 7.91--7.80 (m. 2H),7.70 (dd, J=7.1 and 2.0 Hz, 1H), 7.49 (dd, J=8.6 and 2.0 Hz, 1H), 736(d, J=8.5 Hz, 1H), 3.55 (s, 2H), 3.88 (m, 4H), 2.47 (q, J=7.0 Hz, 4H),2.17 (s, 3H), 0.93 (t, J=7.0 Hz, 6H). Anal Caled. for C₂₂H₂₆N₄O₂: C,69.82; H, 6.92; N 14.80; Pound: C, 69.56; H, 6.95; N, 14.60.

Compound 4i:10-(4-Hydroxy-piperidin-1-ylmethyl-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and Piperidin-4-ol according to generalprocedures B and C. Purification of the product by crystallization fromethanol gave 4 i as a white solid, ¹H-NMR (DMSO-d₆, 300 MHz): 12.60 (s,1H), 7.96 (d, J=1.9 Hz, 1H), 7.91-7.84 (m, 2H), 7.66 (dd, J=6.9 and 2.3Hz, 1H), 7.43 (dd, J=8.6 and 2.1 Hz, 1H), 7.32 (d, J=8.6 Hz, 1H), 4.54(d, J=4.2 Hz, 1H), 3.48 (s, 2H), 3.46 (m, 1H), 2.65 (m, 2H), 2.05 (m,2H), 1.68 (m, 2H), 1.40 (m, 2H). Anal. Calcd. for C₂₀H₁₉N₃O₃: C 68.75;H, 5.48; N, 12.03; Found: C, 68.66; H, 5.48; N, 12.13.

Compound 4j:10-{[Ethyl-(2-hydroxy-ethyl)-amino]-methyl}-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and 2-Ethylamino-ethanol according to generalprocedures B and C. Purification of the product by crystallization fromethanol gave 4j as a white solid. ¹H-NMR (DMSO-d₆, 300 MHz): 1.00 (t,J=6.4 Hz, 3H), 2.45-2.55 (m, 4H), 3.49 (dd, J=12.0 and 5.5 Hz, 2H), 3.64(s, 2H), 4.39 (t, J=5.1 Hz, 1H), 7.35 (d, J=8.5 Hz, 1H), 7.50 (dd, J=8.8and 6.5 Hz, 1H), 7.69 (dd, J=6.5 and 4.3 Hz, 1H), 7.86-7.91 (m, 2H),8.02 0 (d, J=2.0, 1H).

Compound 4k:10-[(Diisopropylamino)-methyl]-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and diisopropylamine according to generalprocedures B and C. Purification of the product by crystallization fromethanol gave 4k as a white solid. ¹H-NMR (DMSO-de₆, 300 MHz): 1.01 (d,J=6.3 Hz, 12H), 2.93-3.04 (m, 2H), 3.66 (s, 2H), 7.34 (d, J=8.4 Hz, 1H),7.51 (dd, J=9.2 and 1.9 Hz, 1H), 7.69 (dd, J=6.5 and 2.7 Hz, 1H),7.86-7.91 (m, 2H), 8.09 (d, J=1.9 Hz, 1H).

Compound 4l:10-(3-Hydroxy-pyrrolidin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and Pyrrolidin-3-ol according to generalprocedures B and C. Purification of the product by crystallization fromethanol gave 4l as a white solid. ¹H-NMR (DMSO-d₆, 300 MHz): 1.50-1.60(m, 1H), 1.95-2.05(m, 1H), 2.31-2.35 (m, 1H), 2.55-2.65 (m, 1H),2.68-2.74 (m, 1H), 3.62 (d, J=4.2 Hz, 2H), 4.18-4.25 (m, 1H), 4.72 (d,J=4.5 Hz, 1H), 7.35 (d, J=8.6 Hz, 1H), 7.46-7.49 (m, 1H), 7.70 (dd,J=6.9 and 2.2 Hz, 1H), 7.87-7.91 (m, 2H), 8.00 (d, J=1.6 Hz, 1H). Anal.Calcd. for C₁₉H₁₇N₃O₃: C, 68.05; H, 5.11; N, 12.53; Found: C, 67.80; H,5.11; N, 12.49.

Compound 4m:10-[4-(2-Hydroxy-ethyl)-piperidin-1-ylmethyl]-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and 2-Piperidin-4-yl-ethanol according togeneral procedures B and C. Purification of the product bycrystallization from ethanol gave 4m as a white solid. ¹H-NMR (DMSO-d₆,300 MHz): 1.12-1.17 (m, 2H), 1.34-1.38 (m, 3H), 1.61 (d, J=12 Hz, 1H),1.92 (t, J=11 Hz, 2H), 2.80 (d, J=10.6 Hz, 2H), 3.42-3.48 (m, 4H), 4.34(t, J=5.2, 1H), 7.33 (d, J=8.8 Hz, 1H), 7.43-7.46 (m, 1H), 7.68 (dd,J=6.9 and 2.5 Hz, 1H), 7.86-7.98 (m, 3H). Anal. Calcd. for C₂₂H₂₃N₃O₃:C, 70.01; H, 6.14; N, 11.13; Found: C, 69.82; H, 6.12; N, 11.08 .

Compound 4n:10-(1,4-Dioxa-8-aza-spiro[4.5]dec-8-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and 1,4-Dioxa-8-aza-spiro[4.5]decane accordingto general procedures B and C. Purification of the product bycrystallization from ethanol gave 4n as a white solid. ¹H-NMR (DMSO-d₆,300 MHz): 1.62 (t, J=5.0, 4H), 2.40-2.50 (m, 4H), 3.55 (s, 2H), 3.85 (s,4H), 7.34 (d, J=9.4 Hz, 1H), 7.47 (d, J=7.5 Hz, 1H), 7.67-7.70 (m, 1H),7.86-7.92 (m, 2H), 7.98 (s, 1H), 12.62 (s, 1H). Anal. Calcd. forC₂₂H₂₁N₃O₄: C, 67.41: H, 5.43; N, 10.98; Found: C, 67.15; H, 5.30; N,11.03.

Compound 4o:10-(3-Hydroxy-piperidin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and piperidin-3-ol according to generalprocedures B and C. Purification of the product by crystallization fromethanol gave 4o as a white solid. ¹H-NMR (DMSO-d₆, 300 MHz): 1.00-1.12(m, 1H), 1.30-1.50 (m, 1H), 1.52-1.95 (m, 4H), 2.66-2.83 (m, 2H),3.45-3.60 (m, 3H), 4.60 (d, J=5.0 Hz, 1H), 7.34-7.48 (m, 2H), 7.68-7.70(dd, J=6.8 and 2.2 Hz, 1H), 7.87-8.00 (m, 3H). Anal. Calcd. forC₂₀H₁₉N₃O₃: C, 68.75; H, 5.48; N, 12.03; Found: C, 68.85; H, 5.48; N,12.10.

Compound 4p:10-(3-Hydroxy-azetidin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and azetidin-3-ol according to generalprocedures B and C. Purification of the product by crystallization fromethanol gave 4p as a white solid. ¹H-NMR (DMSO-d₆, 300 MHz): 2.79 (t,J=6.9 Hz, 2H), 3.51 (t, J=6.3 Hz, 2H), 3.61 (s, 2H), 4.23 (dd, J=12.9and 6.3 Hz, 1H), 5.33 (d, J=6.5 Hz, 1H), 7.33 (d, J=8.4 Hz, 1H),7.71-7.44 (m, 1H), 7.68 (dd, J=6.8, 2.6, 1H), 7.86-7.96 (m, 3H), 12.62(bs, 1H). Anal. Calcd. for C₁₈H₁₅N₃O₃.(0.5 H₂0): C, 65.45; H, 4.88; N,12.72; Found: C, 65.06; H, 4.60; N, 13.03.

Compound 4q:10-[(2-Morpholin-4-yl-ethylamino)-methyl]-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2 and 2-morpholin-4-yl-ethylamine according togeneral procedures B and C. Purification of the product bycrystallization from ethanol gave 4q as a white solid. ¹H-NMR (DMSO-d₆,300 MHz): 2.16 (bs, 1H), 2.34 (bs, 4H), 2.40 (t, J=6.6 Hz, 2H), 2.60 (t,J=6.1 Hz, 2H), 3.56 (t, J=4.6 Hz, 4H), 3.76 (s, 2H), 7.33 (d, J=8.1 Hz,1H), 7.48 (dd, J=8.3 and 1.8 Hz, 1H), 7.68 (dd, J=6.6 and 2.3 , 1H),7.88-7.93 (m, 2H), 8.01 (d, J=1.5, 1H), 12.63 (bs, 1H). Anal. Calcd. forC₂₁H₂₂N₄O₃.(0.75 H₂0): C, 64.35; H, 6.04; N, 14.29; Found: C, 64.35; H,5.91; N, 14.26.

Compound 4r:10-[(4-Hydroxy-cyclohexylamino)-methyl]-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Prepared from compound 2and trans-4-amino-cyclohexanolaccording togeneral procedures B and C. Purification of the product bycrystallization from ethanol gave 4r as a white solid. ¹H-NMR (DMSO-d₆,300 MHz): 1.03-1.16 (m, 4H), 1.75-1.90 (m, 4H), 2.30-2.40 (m, 1H), 3.75(s, 2H), 4.48 (d, J=3.4, 1H), 7.31 (d, J=8.1 Hz, 1H), 7.48 (dd, J=8.5and2.1 Hz, 1H), 7.67 (dd, J=6.8 and 2.4 Hz, 1H,) 7.85-7.92 (m, 2H), 8.02(d, J=1.9 Hz, 1H). Anal. Calcd. for C₂₁H₂₁N₃O₃.(0.5 H₂0).0.05 N₂H₄): C,67.44; H, 5.98; N, 11.61; Found: C, 67.56; H, 5.74; N, 11.68.

Compound 4s:10-(4-Oxo-piperidin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de]anthracen-3-one

Compound 4a (100 mg, 0.24 mmol) was set stirring in acetic acid (3 ml)and to this, at room temperature, was added concentrated HCl (0.6 ml,large excess). The reaction was heated to 90° C. for 1 hour and thencooled to room temperature. Product was isolated by extraction withethyl acetate after basifying to pH 11-12 with 1N NaOH. Organics weredried over magnesium sulfate and concentrated in vacuo to afford 4s as awhite solid (60 mg, 71%). ¹H-NMR (DMSO-d₆, 300 MHz): 2.37 (t, J=2.4 Hz,4H), 2.73 (t, J=2.7 Hz, 4H), 3.69 (s, 2H), 7.38 (d, J=8.5 Hz, 1H), 7.53(dd, J=9.0 and 6.1 Hz), 7.70 (dd, J=7.1 and 4.7 Hz, 1H), 7.86-7.94 (m,2H), 8.06 (d, J=2.4 Hz, 1H).

Other manners, variations or sequences of preparing the compounds of thepresent invention will be readily apparent to those skilled in the art.

The compounds of the present invention may be useful in the free baseform, in the form of base salts where possible, and in the form ofaddition salts, as well as in the free acid form. All these forms arewithin the scope of this invention. In practice, use of the salt formamounts to use of the base form. Pharmaceutically acceptable saltswithin the scope of this invention are those derived from mineral acidssuch as hydrochloric acid and sulfuric acid; and organic acids such asethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, andthe like, giving the hydrochloride, sulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, and the like respectively, orthose derived from bases such as suitable organic and inorganic bases.Examples of pharmaceutically acceptable base addition salts withcompounds of the present invention include organic bases which arenontoxic and strong enough to form such salts. These organic bases andthe use thereof are readily understood by those skilled in the art.Merely for the purpose of illustration, such organic bases may includemono-, di-, and trialkylamines, such as methylamine, diethylamine andtriethylamine; mono-, di-, or trihydroxyalkylamines such as mono-, di-,and triethanolamine; amino acids such as arginine, and lysine;guanidine; N-methylglucosamine; N-methylgiucamine; L-glutamine;N-methylpiperazine; morpho-line; ethylenedianane;N-benzylphenethylamine; tris(hydroxymethyl)antinoethane; and the like.

The acid addition salts of the basic compounds may be prepared bydissolving the free base of the compound of the present invention inaqueous or aqueous alcohol solution or other suitable solventscontaining the appropriate acid or base and isolating the salt byevaporating the solution, or by reacting the free base of the compoundof the present invention with an acid as well as reacting the compoundof the present invention having an acid group thereon with a base suchthat the reactions are in an organic solvent, in which case the saltseparates directly or can be obtained by concentration of the solution.

The compounds of the invention exhibit pharmacological activity and are,therefore, useful as pharmaceuticals. Additionally, the compoundsexhibit central nervous and cardiac vesicular system activity.

PARP Assays

1. IC₅₀

A convenient method to determine IC₅₀ of a PARP inhibitor compound is aPARP assay using purified recombinant human PARP from Trevigan(Gaithersburg, MD), as follows: The PARP enzyme assay is set up on icein a volume of 100 microliters consisting of 100 mM Tris-HCl (pH 8.0), 1mM MgCl₂, 28 mM KCl, 28 mM NaCl, 0.1 mg/ml of DNase I activated herringsperm DNA (Sigma, Mo.), 3.0 micromolar [3H]nicotinamide adeninedinucleotide (470 mci/mmole), 7 micrograms/ml PARP enzyme, and variousconcentrations of the compounds to be tested. The reaction is initiatedby incubating the mixture at 25° C. After 15 minutes of incubation, thereaction is terminated by adding 500 microliters of ice cold 20% (w/v)trichloroacetic acid. The precipitate formed is transferred onto a glassfiber filter (Packard Unifilter-GF/B) and washed three times withethanol. After the filter is dried, the radioactivity is determined byscintillation counting.

The compounds of this invention were found to have potent enzymaticactivity in the range of a few nM to 20 μM in IC₅₀ in this inhibitionassay.

Using the PARP assays described above, approximate IC₅₀ values wereobtained for the following compounds:

TABLE I

R = compound IC₅₀ (uM)

4a 0.03

4b 0.02

4c 0.03

4d 0.02

4f N/A

4e N/A

4g 0.5 

4h 0.1 

4i 0.04

4j 0.1 

4k 0.3 

4l 0.1 

4m 0.1 

4n 0.1 

4o 0.05

4p 0.03

4q 0.05

4r 0.03

4s 0.12

2. Measuring Altered Gene Expression in mRNA Senescent Cells

Gene expression alteration may be measured with human fibroblast BJcells which, at Population Doubling (PDL) 94, are plated in regulargrowth medium and then changed to low serum medium to reflectphysiological conditions described in Linskens, et al., Nucleic AcidsRes., 23, 3244-3251 (1995). A medium of DMEM/199 supplemented with 0.5%bovine calf serum is used. The cells are treated daily for 13 days. Thecontrol cells are treated with and without the solvent used toadminister the PARP inhibitor. The untreated old and young control cellsare tested for comparison. RNA is prepared from the treated and controlcells according to the techniques described in PCT Publication No.96/13610 and Northern blotting is conducted. Probes specific forsenescence-related genes are analyzed, and treated and control cellscompared. In analyzing the results, the lowest level of gene expressionis arbitrarily set at 1 to provide a basis for comparison. Three genesparticularly relevant to age-related changes in the skin are collagen,collagenase and elastin. West, Arch. Derm. 130, 87-95 (1994). Elastinexpression of the cells treated with the PARP inhibitor is expected tobe significantly increased in comparison with the control cells. Elastinexpression should be significantly higher in young cells compared tosenescent cells, and thus treatment with the PARP inhibitor should causeelastin expression levels in senescent cells to change to levels similarto those found in much younger cells. Similarly, a beneficial effectshould be seen in collagenase and collagen expression with treatmentwith the PARP inhibitors.

3. Measuring Altered Gene Expression of Protein in Senescent Cells

Gene expression alteration may be measured with approximately 105 BJcells, at PDL 95-100 which are plated and grown in 15 cm dishes. Thegrowth medium is DMEM/199 supplemented with 10% novice calf serum. Thecells are treated daily for 24 hours with the PARP Inhibitors of (100μg/1 mL of medium). See WO 99/11645. The cells are washed with phosphatebuffered solution (PBS), then permeablized with 4% paraformaldehyde for5 minutes, then washed with PBS, and treated with 100% cold methanol for10 minutes. The methanol is removed and the cells are washed with PBS,and then treated with 10% serum to block nonspecific antibody binding.About 1 mL of the appropriate commercially available antibody solutions(1:500 dilution. Vector) is added to the cells and the mixture incubatedfor 1 hour. The cells are rinsed and washed three times with PBS. Asecondary antibody, goat anti-mouse IgG (1 mL) with a biotin tag isadded along with 1 mL of a solution containing streptavidin conjugatedto alkaline phosphatase and 1 mL of NBT reagent (Vector). The cells arewashed and changes in gene expression are noted colorimetrieally. Foursenescence-specific genes—collagen I, collagen III, collagenase, andinterferon gamma—in senescent cells treated with the PARP inhibitor aremonitored and the results should show a decrease in interferon gammaexpression with no observable change in the expression levels of theother three gens, demonstrating that the PARP inhibitors can altersenescence-specific gene expression.

4. Extending or Increasing Proliferative Capacity and Lifespan of Cells

To demonstrate the effectiveness of the present method for extending theproliferative capacity and lifespan of cells, human fibroblast sellslines (either W138 at Population Doubling (PDL) 23 or BJ cells at PDL71) are thawed and plated on T75 flasks and allowed to grow in normalmedium (DMEM/M199 plus 10% bovine calf serum) for about a week, at whichtime the cells are confluent, and the cultures are therefor ready to besubdivided. At the time of subdivision, the media is aspirated, and thecells rinsed with phosphate buffer saline (PBS) and then trypsinized.The cells are counted with a Coulter counter and plated at a density of10⁵ cells per cm² in 6-well tissue culture plates in DMEM/199 mediumsupplemented with 10% bovine calf serum and varying amounts (0.10 μM,and 1 mM: from a 100× stock solution in DMEM/M199 medium) of a PARPinhibitor. This process is repeated every 7 days until the cells appearto stop dividing. The untreated (control) cells reach senescence andstop dividing after about 40 days in culture.

Administration with Temozolomide Example 1 Oral Administration ofCompound 4i+Temozolomide Enhances Survival of Mice Bearing Malignanciesat the CNS Site

The intracranial transplantation procedure was performed as described inTentori L. et al., “Effects of single or split exposure of leukemiccells to temozolomide, combined with poly(ADP-ribose) polymeraseinhibitors on cell growth, chromosomal aberrations and base excisionrepair components,” Cancer Chemother Pharmacol, 47, 361-9 (2001). Murinemelanoma B16 cells (10⁴) were injected intracranially (ic) into maleB6D2F1 (C578L/6×DBA/2) mice. Histological evaluation of tumor growth inthe brain was performed 1-5 days after tumor challenge, in order todetermine the timing of treatment.

Compound 4i was dissolved in 70 mM PBS without potassium andadministered po 1 h before temozolomide (TMZ). TMZ was dissolved indimethyl-sulfoxide (40 mg/ml), diluted in saline (5 mg/ml) andadministered ip at a dose of 100 mg/Kg for five days. Mice were treatedwith compound 4i by oral gavages once a day for five days, at doses of10 or 40 mg/kg/day. Median survival times (MST) were determined and thepercentage of increase in lifespan (ILS) was calculated as: {[MST (days)of treated mice/MST (days) of control mice]−1}×100. Efficacy oftreatments was evaluated by comparing survival curves between treatedand control groups.

In mice bearing B16 melanoma, the results indicated that the meansurvival time of the groups treated with compound. 4i+TMZ combinationwas significantly higher than that observed in animals receiving TMZ assingle agent. (FIG. I and Table II).

TABLE II Survival rate of mice bearing B16 melanoma in brain TreatmentMST (day) ILS vs TMZ P vs TMZ Control 14 TMZ 100 mg/kg/ip × 5 14Compound 4i po (10 mg/kg) + 17 21 0.001 TMZ × 5 Compound 4i po (40mg/kg) + 21 50 <0.0001 TMZ × 5

Example 2 Administration of Compound 4i Enhances the Effect ofTemozolomide in a Subcutaneous Melanoma Cancer Model

The efficacy of TMZ±Compound 4i treatment was also evaluated on melanomagrowing subcutaneously (s.c.) in mice. For this purpose B16 cells(2.5×10⁵) were inoculated s.c. in the flank of the animal. Tumors weremeasured with calipers and volume calculated according to the formula:[(width)²×length]/2. Drug treatment started 6 days after challenge, whenthe volume of tumor nodules reached 100-150 mm³. Compound 4i (40 mg/kgpo) was administered at 20 min before temozolomide (100 mg/kg ip) once aday for five days. Melanoma growth was monitored by measuring tumornodules every 3 days for 3 weeks.

The combination treatment of Compound 4i+TMZ significantly reduced thegrowth of B16 melanoma (P<0.01 from day 9 to day 23, vs TMZ alone) (FIG.II).

Example 3 Sympathetic Nerve Conductance Velocity (SNCV) inCisplatin-Induced Neuropathy in Rats

The neuroprotective effects of the compounds of the invention weredemonstrated in a model of cisplatin-induced neuropathy in rats. Nerveconduction velocity changes are well documented to be a sensitivemeasure of chemotoxin-induced peripheral neuropathy. Compound 4i wasshown to attenuate the deficits in nerve conduction velocity induced bychronic treatment with cisplatin.

In this experiment, female Wistar Hannover rats were dosed withneuropathy inducing doses of cisplatin (2 mg/kg IP; twice a week for 4weeks) with and without compound 4i (40 mg/kg PO daily). The rats weremonitored for changes in sensory nerve conduction velocity (SNCV) in thecaudal nerve at baseline (pre-cisplatin dosing) and after treatment.Additionally, dorsal root ganglion and sciatic nerve specimens withmorphometric analysis on dorsal root ganglion neurons (somatic, nuclearand nucleolar size) were assessed histopathologically.

At the beginning and end of the treatment period, each animal underwentthe determination of SNCV in the tail as previously described inCavaletti et al., “Protective Effects of glutathione on cisplatinneurotoxicity in rats,” Int. J. Radiation Oncology, 29, 771-776 (1994)and Tredici et al., “Low-Dose Glutathione administration in theprevention of cisplatin-induced peripheral neuropathy in rats,” Neurotoxicology, 15, 701-704 (1994). The antidromic SNCV in the tail nervewas assessed by placing recording ring electrodes distally in the tail,while the stimulating ring electrodes were placed 5 cm and 10 cmproximally with respect to the recording point. The latencies of thepotentials recorded at the 2 sites after nerve stimulation weredetermined (peak-to-peak) and nerve conduction velocity was calculatedaccordingly.

Left L5 dorsal-root ganglia (DRG) of rats from each group were obtainedfrom the sacrificed animals and processed according to previouslyreported protocols [Cavaletti et al.: Tredici et al]], resin embedded,and used for light and electron microscope observations and morphometry.On 1 μm thick semithin sections, morphometric determinations of thecross sectional area of the somata, nuclei and nucleoli of DRG neuronswere performed using an image analysts software (Image J, NIH).

The differences in nerve conduction velocity, and in morphometric dataobtained in dorsal root ganglia neurons during the experiment werestatistically evaluated using the analysis of variance (ANOVA) and theTukey-Kramer post-test (significance level set at p<0.05).

The co-administration of compound 4i was found to induce a statisticallysignificant reduction in the tail nerve conduction velocity impairmentdue to chronic cisplatin treatment (Tables 3 and 4).

TABLE 3 SNCV at the end of the experiment (m/sec) CDDP + controls CDDP4i Number of 6 8 8 values Mean 41.73 28.62 34.08 (m/sec) Std. 1.7180.5194 0.6128 Deviation Std. Error 0.7014 0.1836 0.2166 CDDP = Cisplatin

TABLE 4 Statistical analysis (one-way ANOVA) Tukey's Multiple MeanComparison Test Diff. q P value 95% CI of diff CDDP vs CDDP + 4i −5.46014.90 P < 0.001 −7.016 to −3.905

DRG Morphometry.

A morphometric study on DRG neurons revealed a significant effect onlyon the somatic size of DRG neurons with compound 4i. Table 5 showsmorphometry results, and statistical data is listed in Tables 6 (soma),and 7 (nucleolus).

TABLE 5 Morphometry on DRG (μm²) Soma Nu Nucl Soma Nu Nucl controlscontrols controls CDDP CDDP CDDP Mean 878.3 122.0 9.196 683.4 114.37.651 Std. 547.1 56.46 5.997 376.7 50.05 4.268 Deviation Std. Error23.48 2.423 0.2573 15.89 2.111 0.1800 Soma Nu Nucl CDDP + 4i CDDP + 4iCDDP + 4i Mean 822.1 121.8 8.069 Std. 498.2 55.25 4.587 Deviation Std.Error 20.60 2.284 0.1897 Nu = nucleus, Nucl = nucleolus

TABLE 6 Soma Tukey's Multiple Mean Comparison Test Diff. q P value 95%CI of diff CDDP vs CDDP + 4i −138.7 7.136 P < 0.001 −213.7 to −63.57

TABLE 7 Nucleolus Tukey's Multiple Mean Comparison Test Diff. q P value95% CI of diff CDDP vs CDDP + 4i −0.4177 2.023 P > 0.05 −1.215 to 0.3799

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications are intended to be included within the scope of thefollowing claims.

INCORPORATION BY REFERENCE

All publications, patents, and pre-grant patent application publicationscited in this specification are herein incorporated by reference, andfor any and all purposes, as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. In the case of inconsistencies the presentdisclosure will prevail.

1-26. (canceled)
 27. A combination comprising (i) the compound

 or a pharmaceutically acceptable salt, ester or mixture thereof; and(ii) temozolomide.
 28. The combination of claim 27, wherein the compoundis the maleate salt of


29. A pharmaceutical composition comprising (i) the compound

 or a pharmaceutically acceptable salt, ester or mixture thereof; (ii)temozolomide; and (iii) a pharmaceutically acceptable carrier, diluent,or excipient.
 30. The pharmaceutical composition of claim 29, whereinthe compound is the maleate salt of


31. A method for treating cancer comprising administering an effectiveamount of a combination comprising (i) the compound

 or a pharmaceutically acceptable salt, ester or mixture thereof; and(ii) temozolomide.
 32. The method of claim 31, wherein the compound isthe maleate salt of


33. The method of claim 31, wherein the compound

 is administered in an amount sufficient to potentiate the antitumoractivity of temozolomide.
 34. The method of claim 31, wherein the canceris a tumor.
 35. The method of claim 31, wherein the cancer is selectedfrom melanoma, lymphoma, and glioblastoma multiforme.
 36. The method ofclaim 34 wherein the tumor cells are selected from the group consistingof ACTH-producing tumors, acute lymphocytic leukemia, acutenonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer,brain cancer, breast cancer, cervical cancer, chronic lymphocyticleukemia, chronic myelocytic leukemia, colorectal cancer, cutaneousT-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma,gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin'slymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer(small and/or non-small cell), malignant peritoneal effusion, malignantpleural effusion, melanoma, mesothelioma, multiple myeloma,neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer,ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penilecancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cellcarcinomas, stomach cancer, testicular cancer, thyroid cancer,trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of thevulva and Wilm's tumor.
 37. The method of claim 31, further comprising(a) first: allowing a time period following administration of saidcompound to provide an effective amount of chemosensitization, and, (b)second: administering to said mammal a pharmaceutically-effective doseof temozolomide.
 38. The method of claim 32, further comprising (a)first: allowing a time period following administration of said compoundto provide an effective amount of chemosensitization, and, (b) second:administering to said mammal a pharmaceutically-effective dose oftemozolomide.
 39. The method of claim 31, wherein said compound andtemozolomide are administered essentially simultaneously.
 40. The methodof claim 32, wherein said compound and temozolomide are administeredessentially simultaneously.